Longitudinal evaluation of the biodistribution and cellular internalization of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

Background CD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the tumor uptake and the extracellular dose. To optimize their currently limited efficacy in solid tumors, increased understanding of their pharmacokinetics and in vivo internalization is needed. Methods Here, were studied the pharmacokinetics and in vivo internalization of CD3xTRP1, a fully murine Fc-inert bsAb, in endogenous TRP1-expressing immunocompetent male C57BL/6J mice bearing TRP1-positive and negative tumors over time. Matching bsAbs lacking TRP1-binding or CD3-binding capacity served as controls. BsAbs were radiolabeled with 111In to investigate their pharmacokinetics, target binding, and biodistribution through SPECT/CT imaging and ex vivo biodistribution analyses. Co-injection of 111In- and 125I-labeled bsAb was performed to investigate the in vivo internalization by comparing tissue concentrations of cellular residing 111In versus effluxing 125I. Antitumor therapy effects were evaluated by monitoring tumor growth and immunohistochemistry. Results SPECT/CT and biodistribution analyses showed that CD3xTRP1 specifically targeted TRP1-positive tumors and CD3-rich lymphoid organ and uptake peaked 24 hours pi (KPC3-TRP1: 37.7%ID/g±5.3%ID/g, spleen: 29.0%ID/g±3.9%ID/g). Studies with control bsAbs demonstrated that uptake of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues was primarily receptor-mediated. Together with CD3xTRP1 in the circulation being mainly unattached, this indicates that CD3+ T cells are generally not traffickers of CD3-bsAbs to the tumor. Additionally, target-mediated clearance by TRP1-expressing melanocytes was not observed. We further demonstrated rapid internalization of CD3xTRP1 in KPC3-TRP1 tumors (24 hours pi: 54.9%±2.3% internalized) and CD3-rich tissues (spleen, 24 hours pi: 79.7%±0.9% internalized). Therapeutic effects by CD3xTRP1 were observed for TRP1-positive tumors and consisted of high tumor influx of CD8+ T cells and neutrophils, which corresponded with increased necrosis and growth delay. Conclusions We show that CD3xTRP1 efficiently targets TRP1-positive tumors and CD3-rich tissues primarily through receptor-mediated targeting. We further demonstrate rapid receptor-mediated internalization of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues. Even though this significantly decreases the therapeutical available dose, CD3xTRP1 still induced effective antitumor T-cell responses and inhibited tumor growth. Together, our data on the pharmacokinetics and mechanism of action of CD3xTRP1 pave the way for further optimization of CD3-bsAb therapies.


INTRODUCTION
Immunotherapeutic CD3-targeting bispecific antibodies (CD3-bsAbs) show therapeutic potential in a multitude of malignancies. 1 These bsAbs are designed to specifically bind CD3 and a tumor-associated surface antigen (TAA).On simultaneous binding to CD3 and the TAA, an immunological synapse is formed, resulting in local T-cell activation and tumor-cell killing independent of TCR specificity, and thus elicits antitumor activity even from non-tumor-specific T cells. 2 CD3-bsAbs have shown impressive clinical results for hematological malignancies. 3 4In solid cancers, however, their therapeutic efficacy is still limited for several reasons.First, their immunosuppressive tumor microenvironment is characterized by poor infiltration and reduced effector T-cell function. 5Second, poor vascularization and low or heterogeneous TAA expression may hamper CD3-bsAbs tumor accumulation. 6Third, expression of TAAs on healthy tissues potentially affects pharmacokinetics (PK) and biodistributions of CD3-bsAbs through targetmediated clearance.Furthermore, it increases the potential for adverse effects through "on-target, off-tumor" binding. 7Thus, factors affecting tumor uptake and PK play an important role CD3-bsAbs' efficacy and toxicity profiles, especially for solid tumors. 8Additionally, as the binding of CD3-bsAbs to both the TAA and CD3 on the cell membrane is required to induce the formation of the cytolytic synapse, its extracellular localization is essential for eliciting antitumor T-cell responses.Internalization of CD3-bsAbs consequently negates their therapeutic potential.Therefore, thorough understanding of CD3-bsAbs' PK and in vivo cellular internalization is essential to increase efficacy of novel CD3-bsAbs for solid cancers.
Nuclear imaging of radiolabeled CD3-bsAbs allows for evaluating their PK and biodistribution on a full-body scale.Preclinical models enable detailed translational studies to investigate more complex characteristics, such as in vivo internalization.As preclinical studies with human CD3-bsAbs are restricted to immunodeficient or artificial models because they lack cross-species specificity, they omit the complex immunological interplay of CD3-bsAbs with their targets.Although human CD3 and human TAA knock-in models resolve most of these obstacles, they usually express the proteins at higher levels compared with endogenous expression.Furthermore, their use is only practicable when the TAAs function is not (severely) altered and they might not account for factors such as, steric hinderance. 9Moreover, protection against the generation of anti-IgG antibodies which can affect the human CD3-bsAbs' biodistribution is not guaranteed.Thus, the use of artificial models can considerably complicate the translation of biodistribution results to the clinical setting.Therefore, we investigated the PK, biodistribution, and in vivo internalization of a fully murine Fc-inert bsAb targeting murine CD3ε and murine tyrosinase-related protein 1 (TRP1, also gp75 or TYRP-1) (CD3xTRP1 bsAb) that has been extensively evaluated for its immunological effects. 10 11TRP1 is translationally of interest as it is similarly expressed in mice and humans, with endogenous expression on melanocytes and upregulated expression in melanomas.Here, we report the PK, biodistribution, and in vivo internalization of radiolabeled CD3xTRP1 bsAb and control bsAbs in fully syngeneic tumor mouse models with endogenous TRP1 expression.We show that CD3xTRP1 specifically accumulates in TRP1 expressing tumors and CD3-rich tissues.Furthermore, effective antitumor immune responses were induced, even though rapid bsAb internalization in TRP1 expressing tumors and secondary lymphoid organs occurred.
DTPA-bsAbs were radiolabeled with 111 InCl 3 as described above.Next, unincorporated 111 In was chelated by adding EDTA in phosphate-buffered saline (PBS) to a final 5 mM concentration.Iodine-125 ( 125 I) labeling was performed by incubating DTPA-bsAbs in iodogen coated Eppendorf tubes with Na 125 I (10 -5 M NaOH, Perkin Elmer) in 0.5 M phosphate buffer (pH 7.4) with the total reaction volume adjusted to 100 µL with H 2 O, for 10 min at RT.The reaction was quenched with a saturated tyrosine solution.Labeling efficiencies were evaluated with iTLC as described above and were >95% for in vivo and >90% for in vitro experiments.Labeled products were used without further purification.For in vivo experiments, the reaction mixture was adjusted to pH 7.4 with 10x PBS (pH 7.4).Unlabeled bsAb and sterile pyrogenfree PBS were added to reach a final concentration of 62.5 µg/mL.

In vitro characterization
The purity and stability of the radiolabeled antibodies were assessed with high-performance liquid chromatography (HPLC) and iTLC, respectively.We evaluated whether DTPA-conjugation and radiolabeling affected CD3-or TRP1-binding of CD3xTRP1 and control bsAbs with flow cytometry, lactate dehydrogenase (LDH) cytotoxicity, and in vitro binding assays.

Stability test
The stability of radiolabeled ( 111 In and 125 I) bsAbs was analyzed over time.After radiolabeling (A s : 0.5 MBq/µg), the reaction mixture was purified using a buffer exchange column (PD-10, Cytiva) using PBS supplemented with 0.5% bovine serum albumin (BSA).Next, 1/10 th volume of 1% Tween80 in PBS/0.5%BSAwas added and 160 µL was transferred in triplicate to a lobind Eppendorf tube.Samples were kept at 4°C and iTLC was performed as described above at 0, 24, 72, and 168 hours.

LDH cytotoxicity assay
Proliferation of KPC3 and KPC3-TRP1 was arrested by irradiation with 6000 Rad (Gammacell 1000 Elite, Best Theratronics).Subsequently, 30,000 tumor cells/well were seeded in 96-well plates and allowed to adhere for 4 hours.T cells were isolated from spleens of naïve C57BL/6J mice using nylon wool fiber columns to remove B cells.Then, 300 000 T cells/well and either non-conjugated or DTPA-conjugated CD3xTRP1 bsAbs was added and incubated for 2 days at 37°C.Tumor cell killing was assessed using the CyQUANT LDH cytotoxicity assay (Thermo Fisher) following the manufacturers protocol.Absorbance was measured on a Spectramax iD5 plate-reader (Molecular Devices).The percentage of cytotoxicity was calculated, after correcting for absorbance of medium only and tumor cell only controls, from the absorbance of the treated samples and Triton100-lysed maximum kill control samples.

Animal studies
Male C57BL/6J mice (n=78, 6-8 weeks, Charles River, Germany) were housed under specific-pathogen-free conditions in individually ventilated cages with a filter top (Green line IVC, Tecniplast), in the presence of cage enrichment with food and water ad libitum.For tumor inoculation, mice were injected subcutaneously with 200 µL PBS/0.1%BSAcontaining; (1) 0.8×10 5 KPC3-TRP1 cells (right flank) and 0.8×10 5 KPC3 cells (left flank), or 2) 0.8×10 5 B16F10 cells (right flank).Tumor size was measured using a caliper twice weekly.KPC3 and KPC3-TRP1 tumor-bearing mice were block-randomized based on tumor size and injected with radiolabeled bsAbs 17 days after inoculation.B16F10 tumor-bearing mice received [ 111 In]In-CD3xTRP1 8 days post inoculation.Confounding effects of cage allocation were prevented by pooling mice from different groups.Humane endpoints were based on the general level of discomfort and tumor sizes; animal health was checked daily.Researchers and biotechnicians were blinded for group allocation.

Ex vivo biodistribution analyses
To assess the pharmacokinetics, biodistribution, and therapeutic availability of CD3xTRP1 we performed ex vivo biodistribution experiments with 111 In-and 125 I-labeled CD3xTRP1, CD3xMock, and TRP1xMock in KPC3 and KPC3-TRP1 tumor-bearing mice at several time points.Mice (n=7/group) were injected intraperitoneally in the right caudal abdomen with 200 µL PBS/0.1%BSAcontaining 12.5 µg of a 1:1 mixture of 111 In-and 125 I-labeled; a) CD3xTRP1 ([ 111 In]In and [ 125 I]I, A s : 0.069 and 0.027 MBq/µg, 0.43 and 0.17 MBq, (n=21)), b) CD3xMock (A s : 0.070 and 0.027 MBq/µg, 0.44 and 0.17 MBq, (n=21)), or c) TRP1xMock (A s : 0.069 and 0.027 MBq/µg, 0.43 and 0.17 MBq, (n=21)).At 24, 72, or 168 hours post injection (pi), mice were euthanized by CO 2 / O 2 asphyxiation, blood was collected via heart puncture and relevant tissues (blood, muscle, shaven pigmented skin, shaven non-pigmented skin, right and left inguinal lymph nodes, tumors, spleen, thymus, bone marrow, bone, pancreas, duodenum, heart, lung, kidney, liver, stomach, colon, brown adipose tissue, prostate) were collected for biodistribution analysis.To separate the serum and cell pellet, blood was stored in an Eppendorf tube for 30 min at RT, followed by centrifugation at 2000 g for 10 min at 4°C.Tissue and blood samples were weighed (XPE105DR, Mettler Toledo) and radioactivity concentrations were measured with a well-type gammacounter.Aliquots of injection fluid served as reference controls.Tissue accumulation of [ 111 In]In-bsAb and [ 125 I] I-bsAb was calculated as a percentage of the injected dose (%ID) and normalized for tissue weight (percentage injected dose per gram (%ID/g)).To evaluate the absolute accumulated dose in the tumors, spleen, and lymph nodes, we calculated the %ID accumulated in the entire tissue.In vivo internalization of bsAbs was investigated by evaluating 125 I-to- 111 In ratios of tissue uptake concentrations; on internalization and subsequent degradation 125 I effluxes from the cell, 20 whereas 111 In resides intracellularly. 21Tumor tissues were fixated in PBS/4%formalin for immunohistochemical (IHC) analysis.

Statistical analyses
The required number of animals was determined by a priori sample size calculations for KPC3-TRP1 tumor uptake based on previously obtained data.11 mice were excluded from analysis: (1) one B16F10 tumor did not develop, (2) one B16F10 tumor-bearing and nine KPC3 and KPC3-TRP1 tumor-bearing mice from various groups were misinjected intraperitoneally, which was posteriorly defined as an [ 111 In]In-bsAb blood level ≤1/5th of the groups mean at time of dissection.All results are presented as mean±SD, unless stated otherwise.Differences in [ 111 In]In-bsAbs' blood concentrations and tissue uptake was tested for significance using two-way analysis of variances (ANOVAs) with Bonferroni post hoc tests.Differences in KPC3 and KPC3-TRP1 tumor volumes, and increases of intratumoral CD3 + and CD8 + T cells of CD3xTRP1 or control treated mice were tested for statistical significance using one-way ANOVAs with Bonferroni post hoc tests and two-tailed t-tests, respectively.Statistical significance was defined as p≤0.05 and tests were performed with GraphPad Prism V.9.5.

CD3xTRP1 target binding is unaffected by radiochemical procedures
To assess whether bsAb conjugation with the (radio)metal chelator DTPA and/or radiolabeling affected CD3-or TRP1-binding, we performed flow cytometry, LDH cytotoxicity, and in vitro binding assays.Flow-cytometric analysis of equimolar concentrations showed similar binding of unconjugated CD3xTRP1 and DTPA-CD3xTRP1 to both KPC3-TRP1 and TRP1-expressing B16F10 cells, while no binding was observed to TRP1-negative KPC3 cells (figure 1A).Furthermore, it showed that TRP1 expression levels were approximately fivefold higher on KPC3-TRP1 compared with B16F10 cells.In addition, the LDH cell killing assay showed equal induction of cytotoxicity of naïve splenic T cells to KPC3-TRP1 cells by DTPAconjugated CD3xTRP1 and unconjugated CD3xTRP1 at all tested doses (figure 1B).Moreover, both 111 In-and 125 I-labeled DTPA-CD3xTRP1 showed specific binding to TRP1 and CD3, which was not significantly different from each other (figure 1C).Target binding of [ 111 In] In-CD3xMock to CD3 and [ 111 In]In-TRP1xMock to TRP1 was also confirmed (figure 1D).Stability assays showed minimal release (<1%) of 111 In and 125 I from the radiolabeled antibodies up to 168 hours after radiolabeling (online supplemental figure S2).Taken together, ITC-DTPA conjugation and radiolabeling of CD3xTRP1 did not interfere with target binding and T-cell activation.
CD3xTRP1 distribution is mediated via both the CD3-and TRP1-binding arm We evaluated in vivo target specificity of the CD3-and TRP1-binding arms with SPECT/CT and ex vivo biodistribution studies using 111 In-labeled CD3xTRP1 and control bsAbs lacking either the CD3-binding or TRP1-binding arm.SPECT/CT revealed [ 111 In]In-CD3xTRP1 accumulation in CD3-rich tissues and KPC3-TRP1 tumors and not in parental KPC3 tumors.[ 111 In]In-CD3xMock merely distributed to CD3-rich tissues and not to KPC3-TRP1 tumors (figures 2 and 3), while [ 111 In]In-TRP1xMock uptake was only observed in KPC3-TRP1 tumors.These results indicate that CD3xTRP1 tissue uptake is target mediated.Additionally, SPECT/CT imaging showed accumulation of all three bsAbs in the peri-and extratumoral region extending caudally toward the hip of both KPC3-TRP1 and KPC3 tumors (figures 2 and 3), indicating a non-CD3 and non-TRP1 mediated process.Moreover, in B16F10 tumor-bearing mice this accumulation was not observed, suggesting a tumor model specific process (figure 3A,B).
Biodistribution analyses quantitatively confirmed these results; uptake of [ 111 In]In-TRP1xMock in KPC3-TRP1 tumors was significantly higher compared with [ 111 In] In-CD3xTRP1 (72 hours pi: 74.2%ID/g±15.4%ID/gvs 33.5%ID/g±15.4%ID/g,p<0.001), whereas its uptake in CD3-rich tissues, such as the spleen, was significantly lower (online supplemental figure S2 and table S1).Inversely, uptake of [ 111 In]In-CD3xMock in KPC3-TRP1 tumors was significantly lower compared with bsAbs with a TRP binding arm, while uptake in CD3-rich organs was significantly higher (spleen 72 hours pi, CD3xMock vs CD3xTRP1: 46.4%ID/g±17.7%ID/gvs 23.8%ID/ g±10.4%ID/g,p<0.001).Additionally, this suggests that KPC3-TRP1 and secondary lymphoid organ uptake of CD3xTRP1 was not saturated.Blood levels and marker negative tissue uptake were unaffected by removal of CD3-or TRP1-binding capacity.In pigmented and nonpigmented skin [ 111 In]In-CD3xTRP1 uptake was not statistically significant different.Additionally, it was comparable to skin uptake of CD3xMock and TRP1x-Mock (online supplemental table S1).This indicated no pronounced specific uptake of CD3xTRP1 in healthy Open access pigmented skin.In summary, SPECT/CT imaging and biodistribution analyses showed CD3-specificity and TRP1-specificity of CD3xTRP1.

CD3xTRP1 efficiently targets tumors expressing TRP1
To investigate the in vivo distribution of CD3xTRP1 over time, we evaluated the pharmacokinetics of [ 111 In] In-CD3xTRP1 in C57BL/6J mice bearing: (1) KPC3 and KPC3-TRP1 tumors and (2) B16F10 tumors.CD3xTRP1 blood levels were highest at 24 hours pi and decreased gradually over time (figure 3).KPC3-TRP1 tumor uptake was highest at 24 hours pi (37.7%ID/g±5.3%ID/g)and steadily decreased thereafter (168 hours: 15.1%ID/ g±5.8%ID/g, p<0.001), but was significantly higher compared with KPC3 tumor uptake (168 hours: 3.3%ID/ g±1.5%ID/g, p<0.05).Spleen, lymph node, and bone marrow showed similar trends with CD3xTRP1 uptake peaking 24 hours pi, whereas thymic uptake peaked at 72 hours pi.For a comprehensive overview of the Open access biodistributions see online supplemental table S1.As tumor growth continued after bsAb injection, the weight-corrected uptake concentrations decreased.In terms of absolute tumor uptake (%ID) we observed that CD3xTRP1 in KPC3-TRP1 tumors was constant over time.Thus, the decreased tumor uptake concentrations were likely a result of dilution through tumor growth, accompanied by low additional CD3xTRP1 accumulation (online supplemental table S2).A similar effect was observed for CD3xMock but not for TRP1xMock which continued to accumulate in KPC3-TRP1 tumors over time.SPECT/ CT imaging results of CD3xTRP1 corresponded with the ex vivo biodistribution analyses (figure 3A,C).Additionally, SPECT/CT imaging showed rapid [ 111 In]In-bsAb absorption into the circulation, and uptake in the spleen and KPC3-TRP1 tumor within the first 4 hours following intraperitoneal injection.Lymph node uptake became discernible at 24 hours pi.
To evaluate whether CD3xTRP1 in circulation was bound to circulating CD3 + cells, we determined the localization of [ 111 In]In-CD3xTRP1 in serum and clotted fractions of blood samples over time.All serum fractions contained ~80% of the radiolabel, suggesting that CD3xTRP1 was generally not bound to CD3 + T cells and presumably available for target binding (online supplemental table S5).

CD3xTRP1 undergoes target-mediated internalization
To investigate the availability of tumor-accumulated and tissue-accumulated CD3xTRP1 for interaction with CD3, we assessed the internalization of CD3xTRP1 by co-administering 125 I-and 111 In-labeled bsAbs. 23On bsAb internalization, 125 I is released from the cell, while 111 In is retained intracellularly.Therefore, the 125 I-to- 111 In ratio provides an estimation of how much accumulated bsAb was internalized.In KPC3-TRP1 tumors, [ 125 I]I-C-D3xTRP1 uptake (17.0%ID/g±2.7%ID/g)was significantly lower than [ 111 In]In-CD3xTRP1 uptake at 24 hour pi (37.7%ID/g±5.3%ID/g,p<0.001), resulting in an 125 I/ 111 In ratio of 0.45±0.02,meaning that at least 55% of the accumulated CD3xTRP1 was internalized (figure 4, online supplemental table S3 and 4).Significant internalization continued thereafter resulting in approximately 80% of KPC3-TRP1 tumor-accumulated CD3xTRP1 to be unavailable for CD3 interaction at both 72 and 168 hours pi ( 125 I/ 111 In-ratios: 0.22±0.03and 0.21±0.03).In CD3-rich tissues internalization occurred even faster with 80% of spleen and lymph node accumulated CD3xTRP1 being internalized at 24 hours pi (spleen 125 I/ 111 In-ratio: 0.20±0.01).In blood and marker negative tissues, the In accordance with its targets, CD3xMock internalization in CD3-rich tissues was similarly high as CD3xTRP1 (online supplemental table S4), whereas the 125 I/ 111 In ratios of CD3xMock were comparable in KPC3 and KPC3-TRP1 tumors.Likewise, TRP1xMock internalization in KPC3-TRP1 tumors was similarly high when compared with CD3xTRP1, but considerably lower in CD3-rich tissues.This indicated that internalization of CD3xTRP1 in KPC3-TRP1 tumors and CD3-rich tissues were mostly TRP1-or CD3-mediated.Non-target mediated internalization, however, also occurred as the 125 I/ 111 In ratios of CD3xTRP1 and control bsAbs decreased over time in TRP1-negative KPC3 tumors.Additionally, the 125 I/ 111 In ratio of TRP1xMock decreased over time in secondary lymphoid organs, indicating internalization through non-specific processes (online supplemental table S4).In summary, CD3xTRP1 rapidly internalized in vivo in KPC3-TRP1 and CD3-rich tissues, this was mostly target-mediated.

CD3xTRP1 reduces KPC3-TRP1 tumor growth and induces immune-cell infiltration
We assessed the therapy effects of a single injection of radiolabeled CD3xTRP1 and control bsAbs by measuring tumor sizes and evaluating the tumor microenvironment for CD3, CD8, Ly6G, F4/80, and PD-L1.KPC3-TRP1 tumors were significantly smaller in mice treated with CD3xTRP1 compared with TRP1xMock at 168 hours pi (mean±SD: 408±170 vs 751±246 mm 3 , p<0.05) (figure 5A), whereas KPC3 tumor volumes showed no statistically significant differences.Immunohistochemistry revealed substantial increases in necrotic tumor surface area in CD3xTRP1 treated KPC3-TRP1 tumors.This corresponded with increased CD3 + and CD8 + T-cell numbers in KPC3-TRP1 tumors, as determined by the proportion of CD3-stained or CD8-stained tumor area, which was  111 In-and 125 I-labeled CD3xTRP1, at 24, 72, and 168 hours post intraperitoneal injection.Blood (A), spleen (B), KPC3 (C), and KPC3-TRP1 (D) tumor uptake are given as %ID/g, differences in uptake were tested for significance using two-way ANOVAs with a Bonferroni post hoc test (***p<0.001).ANOVA, analysis of variance; %ID, percentage of the injected dose.

Sandker GGW, et al. J Immunother Cancer
Open access most pronounced at 72 hours pi (figure 5B,C, KPC3 vs KPC3-TRP1: 0.11±0.02vs 5.45±0.95%CD3-DAB-stained area, p<0.01).Furthermore, we observed an increase of neutrophils (Ly6G pos ) and a shift from a heterogeneous to more homogeneous intratumoral neutrophil distribution in vital KPC3-TRP1 tumor regions (online supplemental figure S4).Additionally, high numbers of Ly6G pos neutrophils and PD-L1 pos cells were observed in necrotic areas.Moreover, F4/80 high macrophages surrounded the tumor tissue, while tumor infiltrated macrophages appeared to be F4/80 low .The number and intratumoral distribution of both F4/80 high and F4/80 low macrophages did not change over time.Finally, CD3xTRP1 markedly increased membranous PD-L1 expression on KPC3-TRP1 cells on all evaluated time points but most pronounced at 24 and 72 hours pi.
In CD3xMock treated KPC3-TRP1 tumors, the observed effects were substantially less pronounced, with marginally increased necrotic tumor areas, CD8 + T cell numbers, and PD-L1 tumor cell expression only at 24 and 72 hours pi (online supplemental figure S5).In TRP1xMock treated mice all IHC evaluated features were comparable for KPC3-TRP1 and KPC3 tumors.In none of the KPC3 tumors, the effects observed in KPC3-TRP1 tumors were present.In summary, CD3xTRP1 effectuated antitumor immune responses that were predominantly mediated by CD8 + lymphocytes and resulted in necrosis, transient upregulation of PD-L1 on tumor cells, and growth delay in KPC3-TRP1 tumors.

DISCUSSION
Immunotherapeutic CD3-targeting bispecific antibodies cross-link tumor cells with cytotoxic T cells, irrespective of their TCR specificity, thereby inducing local antitumor T-cell responses. 2To further optimize their efficacy, it is important to understand their pharmacokinetics, uptake in tumor and normal tissues, and cellular internalization.Here, we evaluated these aspects in vivo using a completely syngeneic mouse model consisting of a full-length radiolabeled murine CD3xTRP1 bsAb, immunocompetent mice which endogenously express the TAA, and murine TRP1expressing tumors.We demonstrate that CD3xTRP1 distributes to TRP1-positive tumors and CD3-positive tissues, and that a large fraction of CD3xTRP1 is internalized.Nonetheless, antitumor immune responses were induced as observed by profound CD8 + T-cell influx and delayed tumor growth.
In vivo pharmacokinetic SPECT/CT imaging and ex vivo biodistribution studies showed that on intraperitoneal administration, CD3xTRP1 is readily absorbed into the circulation, and distributes specifically toward KPC3-TRP1 tumors and CD3-rich tissues, with uptake concentrations peaking 24 hours pi.The following decrease in tumor uptake concentrations was most likely an effect of increasing tumor volume as the absolute tumor uptake, uncorrected for tumor weight, remained constant.In a clinically more relevant tumor model B16F10, with endogenous TRP1 expression levels at approximately fivefold lower than KPC3-TRP1, 10 the tumor uptake of CD3xTRP1 was only twice as low, whereas the overall PK and biodistribution were similar.Although biological differences in tumors, such as vasculature, permeability, and interstitial pressure might differentially affect CD3xTRP1 uptake in these tumor models, these data suggest that TAA expression levels influence the absolute tumor-targeted dose, but not necessarily in a one-to-one correlation.
Our studies with the control bsAb lacking CD3-affinity, together with the initial absence of tumor-residing CD3 + T cells, shows that uptake of CD3xTRP1 in TRP1-positive tumors is primarily TRP1-mediated.It should be noted that CD3xTRP1 treatment resulted in lower KPC3-TRP1 tumor weight compared with control bsAb treatment, which will impact weight-normalized tumor uptake values (%ID/g).However, analyses of the non-normalized data (%ID), did not change the outcome of the study.Circulatory CD3xTRP1 predominantly resided in the serum fraction of blood, indicating that CD3 + T cells are generally not traffickers of CD3-bsAbs to the tumor.In pigmented skin, where endogenous TRP1 is expressed by melanocytes, no measurable specific accumulation of CD3xTRP1 was observed.A potential explanation might be that the localization of melanocytes in the avascular epidermis hinders efficient targeting of CD3xTRP1. 24This indicates that "on-target, off-tumor" targeting to endogenously expressed TAA does not necessarily affect biodistributions through target-mediated clearance for this bsAb.
Overall, CD3xTRP1's biodistribution is comparable with other CD3-bsAbs that primarily show uptake in lymphoid organs and TAA-expressing tissues. 25 26For example, in knock-in mice engineered to express human CD3 and the membrane-proximal region of MUC16, the human antibody 89 Zr-DFO-REGN4018 recognizing these epitopes showed a highly similar biodistribution pattern compared with our study. 9However, the uptake of CD3xTRP1 in each organ is consistently lower, which might be explained by variations in the bsAbs affinities, which were undisclosed for REGN4018.Nonetheless, they also showed minimal "on-target, off-tumor" targeting and concluded that the targeting toward the tumor is mainly TAA mediated.This is supportive of the translatability of our findings to the clinical setting.Furthermore, as expected, target-tissue uptake is significantly higher compared with molecules with considerably lower circulatory half-lives, such as bispecific T-cell engagers. 27 28PECT/CT imaging further revealed peritumoral uptake of CD3xTRP1 and control bsAbs around both KPC3 and KPC3-TRP1 but not B16F10 tumors, indicating that it was tumor-type specific and not TRP1-or CD3mediated.As the Fc-portion of CD3xTRP1 was "LALA" mutated to minimize Fcγ-receptor (FcγR) binding, an FcγR-mediated uptake is also unlikely.However, to definitively exclude FcγRs' involvement a "PGLALA" mutation could be introduced. 29Therefore, potential explanations for the non-specific uptake include increased angiogenesis Open access and/or leakiness in the peri-tumoral area, trapping in extracellular matrix deposits induced by tumor cells, or accumulation in extratumoral exudate excreted by the stroma and/or KPC3 cells of pancreatic ductal origin. 14 30o exert their therapeutic effect, CD3-bsAbs require simultaneous binding of TAA and CD3 on the membrane of tumor and CD3 + T cells, respectively.Therefore, internalization inevitably decreases its therapeutical available dose.Interestingly, for the therapeutically active CD3xTRP1, we observed receptor-mediated internalization in both KPC3-TRP1 tumors and CD3-rich tissues.In the tumor, this decreased the extracellular dose by half within 24 hours and to ~1/5th within 72 hours pi.In secondary lymphoid organs, even faster internalization resulted in endocytosis of 80% of spleen accumulated CD3xTRP1 within 24 hours.This may be explained by endocytosis signals in CD3ε subunits, which is normally involved in constitutive recycling and downregulating expression of CD3ε, which can be triggered by T-cell activation and antibody binding. 31 32Additionally, we observed non-target-mediated internalization of CD3xTRP1, CD3xMock, and TRP1xMock in KPC3 tumors and secondary lymphoid organs, although considerably slower.Possible mechanisms include micropinocytosis, which occurs in T cells and pancreatic ductal adenocarcinomas, 33 34 and phagocytosis of tumor or immune-cell debris by tumor-infiltrated macrophages.Others have also observed internalization of CD3-bsAbs in tumors and secondary lymphoid organs at a fixed 72 hours pi time point using a CD3-bsAb targeting human-CD3ε in transgenic mice expressing both mouse and human CD3ε. 25e expand on that knowledge by providing a longitudinal overview of this process with our fully functional mouse CD3-bsAb in a non-artificial setting.Previously, on administering CD3xTRP1, transient T-cell activation was observed throughout the body, except for those in the tumor, which stayed activated for at least 4 days. 10This might be explained by the higher internalization rate in secondary lymphoid organs compared with KPC3-TRP1 tumors, which suggests that balancing CD3-bsAb internalization rates can help mitigate immune related adverse effects (IRAEs), while still inducing prolonged activation of antitumor T-cell responses.
Despite rapidly internalizing, CD3xTRP1 managed to elicit antitumor immune responses as KPC3-TRP1 tumors showed delayed growth, influx of CD8 + T cells, increases in necrotic areas, and a subsequent influx of neutrophils.Thus, the rapidly decreasing non-internalized CD3xTRP1 fraction was apparently sufficient for a therapeutic response.These results correspond with previous findings for CD3xTRP1. 10Additionally, we observed CD3xTRP1mediated upregulation of the immune checkpoint PD-L1 on TRP1-positive tumors, which is probably an effect of interferons produced by activated immune cells.[37] Although CD3xTRP1 shows favorable distribution characteristics resulting in effective antitumor immune responses, this study does show opportunities for improving CD3-bsAb immunotherapy by (1) increasing tumor uptake and (2) reducing internalization rates.To increase absolute CD3xTRP1 tumor uptake, shifting uptake from secondary lymphoid organs toward the tumor is preferred above increasing the administered dose as both the tumor and secondary lymphoid organs uptake were not saturated.For high TAA-expressing tumors (eg, KPC3-TRP1) this can be accomplished by decreasing the CD3-affinity of the bsAb, whereas for low TAA expressing-tumors the resulting decrease in therapeutic potency can prohibit this approach. 26 37Additionally, lowering CD3-affinities may reduce CD3-bsAbs' IRAE potential. 26 38Other methods to increase tumor uptake include using engineered antibodies formats, such as asymmetric "2-to-1" bsAbs, or using masked CD3binding arms which are unmasked within the tumor to prevent "on-target, off-tumor" binding. 39This can also limit activation-induced cell death in secondary lymphoid organs through Abs:CD3-binding, thereby increasing the number of healthy non-anergic T cells that can be recruited for antitumor responses. 40A second approach to improve CD3-bsAb immunotherapy is through reducing internalization rates.In the tumor, reduced internalization increases the extracellular available dose, thereby mitigating the loss of therapeutic efficacy.This may be accomplished using endocytosis inhibitors, which are currently being developed. 41 42Another option may be to reduce TAA affinity, as it has been shown that internalization rate increases with epitope affinity for other targets, although contradicting studies exist. 43In secondary lymphoid organs, when not subsequently retained through subsite rebinding, reduced internalization could result in an increase in CD3-bsAb in circulation, which consequently becomes available for tumor targeting.
Given the many different CD3-bsAb formats that have been developed, which all have different TAA targets, PKs, biodistribution, internalization characteristics, and dosing schedules, it is difficult to provide a clear generalization of our findings to these different formats. 25 26 44Moreover, secondary lymphoid organs are easily accessible and express large amounts of CD3, therefore they first require target saturation resulting in non-linear pharmacokinetics of CD3-bsAbs.As we investigated a previously determined optimal therapeutic dose (12.5 µg 10 ) other doses and effective doses of other CD3-bsAbs might result in different biodistributions, as this is partly determined by the administered dose.All in all, we recommend using preclinical nuclear imaging for clinical CD3-bsAb candidates to obtain insights into key characteristics, such as relative tumor and secondary lymphoid organs targeting and in vivo internalization potency.For studying these human CD3-bsAbs, the use of syngeneic models is preferred to avoid effects such as, transfectioninduced differences in TAA expression levels.When artificial models are unavoidable, models closely mimicking